RESUMO
OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
Assuntos
Sobrevivência Celular , Difosfonatos , Endossomos , Fibroblastos , Gengiva , Hidrazonas , Imidazóis , Ácido Zoledrônico , Ácido Zoledrônico/farmacologia , Humanos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/citologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Hidrazonas/farmacologia , Células Cultivadas , Fatores de Tempo , Reação em Cadeia da Polimerase em Tempo Real , Conservadores da Densidade Óssea/farmacologia , Reprodutibilidade dos Testes , Microscopia Confocal , Relação Dose-Resposta a Droga , Pinocitose/efeitos dos fármacosRESUMO
New donor-acceptor alternating conjugated polymers were synthesized and characterized. Among them, PCPBBT exhibited a band-gap of 1.01 eV and ambipolar characteristics with µ(h) = 7.1 × 10(-4) cm(2) V(-1) s(-1) and µ(e) = 3.3 × 10(-3) cm(2) V(-1) s(-1).